This invention relates to a process for producing a complex carbohydrate which is useful for protection against infection of bacteria, viruses and the like, application to cardiovascular disorders and immunotherapy and to a process for producing a sugar nucleotide which is important as a substrate for the synthesis of the complex carbohydrate.
Examples of the known process for producing sugar nucleotides includes: 1) chemical synthetic processes (Adv. Carbohydr. Chem. Biochem., 28, 307 (1973), Bull. Chem. Soc. Japan, 46, 3275 (1973), J. Org. Chem., 57, 146 (1992), Carbohydr. Res., 242, 69 (1993)); 2) production processes using enzymes (J. Org. Chem., 55, 1834 (1990), J. Org. Chem., 57, 152 (1992), J. Am. Chem. Soc., 110, 7159 (1988), Japanese Published Unexamined National Publication No. 508413/95, Japanese Published National Publication No. 500248/95, WO 96/27670); 3) processes using microbial cells such as yeast and the like (Japanese Published Examined Patent Application No. 2073/70, Japanese Published Examined Patent Application No. 40756/71, Japanese Published Examined Patent Application No. 1837/72, Japanese Published Examined Patent Application No. 26703/72, Japanese Published Examined Patent Application No. 8278/74, Japanese Published Unexamined Patent Application No. 268692/90); and 4) an extraction process from microbial cells of halo-tolerant yeast (Japanese Published Unexamined Patent Application No. 23993/96).
However, the process 1) requires expensive materials (for example, morpholidate derivative of nucleotide-5xe2x80x2-monophosphate (referred to as xe2x80x9cNMPxe2x80x9d hereinafter), sugar phosphate, etc.); the process 2) requires expensive materials (for example, nucleotide-5xe2x80x2-diphosphate (referred to as xe2x80x9cNDPxe2x80x9d hereinafter), nucleotide-5xe2x80x2-triphosphate (referred to as xe2x80x9cNTPxe2x80x9d hereinafter), phosphoenolpyruvate, etc.), and various enzymes (e.g., pyruvate kinase, etc.); and the process 3) requires drying treatment of microbial cells. Including the process 4), all of the above-mentioned processes use expensive nucleotides, sugar phosphates, and the like or have a difficulty in affecting large scale production from the operational point of view, so that an industrial scale production process of sugar nucleotides has not so far been established.
Examples of the known process for producing complex carbohydrates include 1) chemical synthetic processes (Method in Enzymol., 247, 193 (1994), Angew. Chem. Int. Ed. Engl., 21, 155 (1982), Carbohydr. Res., 211, cl (1991)), 2) processes in which a hydrolase is used (Anal. Biochem., 202, 215 (1992), Trends Biotechnol., 6, 256 (1988)) and 3) processes in which a glycosyltransferase is used (Japanese Published Unexamined Patent Application No. 79792/95, Japanese Published National Publication No. 500248/95, Japanese Published Examined Patent Application No. 82200/93, WO 94/25614, Japanese Published National Publication No. 503905/97, U.S. Pat. No. 5,583,042).
The introduction of protecting groups is essential for stereo-selective synthesis in the process 1). The yield and selectivity are not sufficient in the process 2). Expensive materials (for example, NDP, NTP, phosphoenolpyruvic acid, sugar phosphate, sugar nucleotide, etc.) and various enzymes (for example, pyruvate kinase, etc.) are necessary in the process 3). Therefore, these processes have not been established as inexpensive industrial production processes of complex carbohydrates. In addition, there has been nothing known about a process for the direct industrial production of complex carbohydrates, which uses only inexpensive nucleotide precursors, sugars and complex carbohydrate precursors as the starting materials.
It has been reported that UMP is produced in a microorganism belonging to the genus Corynebacterium when orotic acid is added (Amino Acid, Nucleic Acid, 23, 107(1971)). In addition, a process in which cytidine diphosphate choline is formed from orotic acid as the material is also known (Japanese Published Unexamined Patent Application No. 276974/93).
An object of the present invention is to provide a process for producing a complex carbohydrate which is useful for protection against infection of bacteria, viruses, and the like, application to cardiovascular disorders and immunotherapy, and a process for producing a sugar nucleotide which is important as a substrate for synthesizing the complex carbohydrate at a low cost and efficiently.
The inventors of the present invention have conducted intensive studies on the microbial production of a complex carbohydrate and a sugar nucleotide using a nucleotide precursor as the starting material, and have found as the results that a sugar nucleotide can be produced by using only a nucleotide precursor and a sugar as the materials, that productivity of the sugar nucleotide can be improved by strengthening expression of genes involved in its biosynthesis and that a complex carbohydrate can be produced efficiently, using a microorganism capable of producing the sugar nucleotide and a microorganism, an animal cell or an insect cell capable of producing the complex carbohydrate from a sugar nucleotide and a complex carbohydrate precursor and using a nucleotide precursor, a sugar and a complex carbohydrate precursor as the only starting materials, thereby resulting in the accomplishment of the present invention.
The present invention provides a process for producing a complex carbohydrate, which comprises: selecting, as enzyme sources, a) a culture broth of a microorganism capable of producing NTP from a nucleotide precursor, or a treated product of the culture broth, b) a culture broth of a microorganism capable of producing a sugar nucleotide from a sugar and NTP, or a treated product of the culture broth, and c) a culture broth of a microorganism, an animal cell or an insect cell capable of producing a complex carbohydrate from a sugar nucleotide and a complex carbohydrate precursor, or a treated product of the culture broth; allowing the enzyme sources, the nucleotide precursor, the sugar and the complex carbohydrate precursor to be present in an aqueous medium to form and accumulate the complex carbohydrate in the aqueous medium; and recovering the complex carbohydrate from the aqueous medium, a process for producing a sugar nucleotide, which comprises: selecting, as enzyme sources, a) a culture broth of a microoganism capable of producing NTP from a nucleotide precursor, or a treated product of the culture broth, and b) a culture broth of a microorganism capable of producing a sugar nucleotide from a sugar and NTP, or a treated product of the culture broth; allowing the enzyme sources, the nucleotide precursor and the sugar to be present in an aqueous medium to form and accumulate the sugar nucleotide in the aqueous medium; and recovering the sugar nucleotide from the aqueous medium, and a process for producing a complex carbohydrate, which comprises: selecting, as an enzyme source, a culture bloth of a microorganism, an animal cell or an insect cell capable of producing a complex carbohydrate from a sugar nucleotide and a complex carbohydrate precursor, or a treated product of the culture broth; allowing the enzyme source, the sugar nucleotide obtained by the above-described process for producing a sugar nucleotide and the complex carbohydrate precursor to be present in and aqueous medium to form and accumulate the complex carbohydrate in the aqueous medium; and recovering the complex carbohydrate from the aqueous medium. It also provides a process for producing N-acetylglucosamine-1-phosphate, which comprises selecting, as an enzyme source, a culture broth of a microorganism having strong galactokinase activity, or a treated product of the culture broth; allowing the enzyme source and N-acetylglucosamine to be present in an aqueous medium to form and accumulate N-acetylglucosamine-1-phosphate in the aqueous medium; and recovering the N-acetylglucosamine 1-phosphate from the aqueous medium.